Antiproliferative effects of Plumbago rosea and its purified constituent plumbagin on SK-MEL 28 melanoma cell lines.

BACKGROUND: Plumbago rosea is used in traditional systems of medicine for the preparation of formulations used for treating inflammations, cough, bronchitis, and gastrointestinal disorders, and also in conjunction with cancer chemotherapy. In the present study, the cytotoxic and anti-proliferative effects of plumbagin, and the ethanolic root extract of P. rosea (ETPR) was evaluated on SK-MEL 28 melanoma cell lines and human lymphocytes. MATERIALS AND METHODS: MTT and apoptotic assays were used for the evaluation of cytotoxic and anti-proliferative effects, respectively. In addition, the effect of Plumbagin and ETPR in down regulation of BCL-2 expression is investigated using RT-PCR analysis. RESULTS: Both plumbagin and ETPR dose-dependently decreased the cell viability more potently in melanoma cell lines. P. rosea extract demonstrated significant synergy in inhibiting BCL-2 expression than plumbagin. Moreover plumbagin showed more toxicity in human lymphocytes. CONCLUSION: Plumbagin has anti-cancer potential, but the side effects limits its use; yet plumbagin, in combination with other ingredients in Plumbago rosea extract, displays significant synergy leading to a stronger anticancer effect with significantly less toxicity.

Mutual adaptation between mouse transglutaminase 4 and its native substrates in the formation of copulatory plug.

Formation of copulatory plugs by male animals is a common means of reducing competition with rival males. In mice, copulatory plugs are formed by the coagulation of seminal vesicle secretion (SVS), which is a very viscous and self-clotting fluid containing high concentration of proteins. In its native state, mouse SVS contains a variety of disulfide-linked high-molecular-weight complexes (HMWCs) composed of mouse SVS I-III, which are the major components of mouse SVS. Further, mouse SVS I-III are the substrates for transglutaminase 4 (TGM4), a cross-linking enzyme secreted from the anterior prostate. According to activity assays, mouse TGM4 prefers a mild reducing and alkaline environment. However, under these conditions, the activity of mouse TGM4 toward SVS I-III was much lower than that of a common tissue-type TGM, TGM2. On the other hand, mouse TGM4 exhibited much higher cross-linking activity than TGM2 when native HMWCs containing SVS I-III were used as substrates under non-reducing condition. By the action of TGM4, the clot of SVS became more resistant to proteolysis. This indicates that the activity of TGM4 can further rigidify the copulatory plug and extend its presence in the female reproductive tract. Together with the properties of TGM4 and the nature of its disulfide-linked SVS protein substrates, male mice can easily transform the semen into a rigid and durable copulatory plug, which is an important advantage in sperm competition.

Identification of the major TG4 cross-linking sites in the androgen-dependent SVS I exclusively expressed in mouse seminal vesicle.

SVS I was exclusively expressed in seminal vesicle in which the protein was immunolocalized primarily to the luminal epithelium of mucosal folds. The developmental profile of its mRNA expression was shown to be androgen-dependent, manifesting a positive correlation with the animal's maturation. There are 43 glutamine and 43 lysine residues in one molecule of SVS I, which is one of the seven major monomer proteins tentatively assigned on reducing SDS-PAGE during the resolution of mouse seminal vesicle secretion. Based on the fact that SVS I-deduced protein sequence consists of 796 amino acid residues, we produced 7 recombinant polypeptide fragments including residues 1-78/F1, residues 79-259/F2, residues 260-405/F3, residues 406-500/F4, residues 501-650/F5, residues 651-715/F6, and residues 716-796/F7, and measured the covalent incorporation of 5-(biotinamido)pentylamine (BPNH(2)) or biotin-TVQQEL (A25 peptide) to each of F1-to-F7 by type 4 transglutaminase (TG(4)) from the coagulating gland secretion. F2 was active to a greater extent than the other fragments during the BPNH(2)-glutamine incorporation, and a relatively low extent of A25-lysine cross link was observed with all of the seven fragments. The MS analysis of BPNH(2)-F2 conjugate identified Q(232) and Q(254) as the two major TG(4) cross-linking sites. This was substantiated by the result that much less BPNH(2) was cross-linked to any one of the three F2 mutants, including Q232G and Q254G obtained from single-site mutation, and Q232G/Q254G from double-site mutation.